Transformation of breast cells by truncated neurokinin-1 receptor is secondary to activation by preprotachykinin-A peptides

Abstract

Breast cancer remains the cancer with the highest mortality among women in the United States. Peptides derived from the oncogenic Tac1 gene (full transcript: betaPPT-A) stimulate the proliferation of breast cancer cells (BCCs) via seven-transmembrane G protein-coupled neurokinin 1 (NK1) and NK2 receptors. The NK1 gene could generate full-length (NK1-FL) and truncated (NK1-Tr) transcripts. NK1-Tr lacks 100 residues in their cytoplasmic end, could couple to G proteins, and shows reduced efficiency with respect to internalization and desensitization. This study reports on a role of NK1-Tr in the transformation of nontumorigenic breast cells, and investigates whether Tac1 expression is linked to the generation of NK1-Tr. Western blots and Northern analyses showed coexpressions of NK1-Tr and NK1-FL in BCCs (cell lines and primary cells from patients with different stages of breast cancer). Stable transfections of betaPPT-A or NK1-Tr expression vectors in nontumorigenic cells showed each induces the expression of the other, consequently resulting in a transformed phenotype. Analyses with microarrays indicate similar patterns of cytokine production by NK1-Tr transfectants and BCCs, but not NK1-FL transfectants. These observations indicate tumor-promoting properties by NK1-Tr, but not NK1-FL. Overall, the oncogenic property of Tac1 in breast cells involves concomitant expression of NK1-Tr and vice versa, consequently leading to the production of cytokines with growth promoting functions.

Fig. 1.

Coexpression of NK1-FL and NK1-Tr cDNA in BCCs. Membrane extracts were examined for NK1 by Western blots in BCCs (A); nontumorigenic breast cell lines (B), and primary BCCs, with DU4475 as control (C).

Fig. 2.

Stable transfection of NK1-Tr and NK1-FL cDNA in MCF12A and MCF10A. (A) The mean fluorescence intensities (MFI, mean ± SD) are shown for transfectants for five cell passages. (B) Substance P production (mean ± SD, n = 10) in the culture media of NK1-FL and NK1-Tr transfectants. (C) Representative confluent cultures for NK1 transfectants. ^*, P < 0.05 vs. untransfected and NK1-FL transfectants.

Fig. 3.

Growths curves and clonogenic assays for nontumorigenic cell lines. MCF10A and MCF12A, untransfected or stably transfected with NK1-Tr (A) or NK1-FL (B) in the sense and antisense orientations. Cells were seeded at 100 per ml in 2 ml of culture media. Transfectants with NK1-Tr cDNA were cultured in the presence or absence of NK1-specific antagonist (10 nM C-99,994). Beginning at day 1, viable cells were counted daily up to day 5. The mean ± SD, n = 10 represent five cell passages per transfectant. (C) Clonogenic assays were performed with cells from five cell passages at 10³ cells per ml. At day 5, colonies with >20 cells were counted, and the results were presented as the mean ± SD, n = 10. ^*, P < 0.05 vs. sense transfectants; ^**, P < 0.05 vs. sense transfectants; ^***, P < 0.05 vs. NK1-FL transfectants and untransfected/antisense.

Fig. 4.

NK1 expression in PPT-A knockdown BCCs and βPPT-A expressing nontumorigenic breast cells (MCF12A, MCF10A, MCF12F, MCF10-2A). Northern analyses for NK1-FL and NK1-Tr mRNA were determined in βPPT-A-expressing cells. (A) Representative untransfected nontumorigenic cell is shown for MCF12A in the left lane. (B) Tac1 knockdown in T47D and BT474 with pPMSKH1-PPT-A. In the left lane, control transfectants with pPMSKH1 alone shown for T47D. (C) The density of NK1-FL is normalized to 10, and fold changes in PPT-A knockdown BCCs are shown for the six breast cancer cell lines described in Materials and Methods. Western blots with anti-NK-1 using membrane extracts from two breast cancer cell lines and two patients are shown. Arrows represent transfectants with pPMSKH1-PPT-A compared to transfectants with pPMSKH1 alone (no arrow).